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T7 DNA Polymerase with fluorescent unnatural amino acid

ABSTRACT: A T7 DNA polymerase variant with the fluorescent unnatural amino acid (7-hydroxycoumarin-4-yl) ethylglycine (7-HCou) incorporated at position 514 was expressed in E. coli and purified. The protein was then characterized by mass spec, pre-steady state kinetic experiments, and fluorescence measurements.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Escherichia Virus T7

SUBMITTER: Tyler Dangerfield

LAB HEAD: Kenneth A. Johnson

PROVIDER: PXD021676 | Pride | 2020-10-19

REPOSITORIES: Pride

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Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication.

Dangerfield Tyler L TL Johnson Kenneth A KA

The Journal of biological chemistry 20201005 50

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli MS ...[more]

PMID: 33020184

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